Apoptosis is a regulated form of cell death that is necessary for normal cell function and development. The regulation of apoptosis in a cell is mediated by diverse signals and complex interactions of many different cellular gene products. Dysregulation of apoptosis can cause or contribute to a variety of diseases and conditions. However, having the ability to induce apoptosis in target cells in certain disease states would be extremely advantageous. Over the past several years, numerous gene products which modulate the apoptotic process have been identified. The discovery of such gene products presents opportunities to intervene in normal and abnormal cellular processes and regulate cell death for therapeutic purposes.
Fas is one such gene product. Fas (CD95/APO-1) is a transmembrane glycoprotein that is related to the receptors for tumor necrosis factor and nerve growth factor (Itoh et al., 1991, Cell 66:23-243; Oehm et al., 1992, J. Biol. Chem. 267:10709-10715). Upon being cross-linked with agonistic anti-Fas antibodies or Fas ligand (FasL), Fas initiates a complex signal transduction pathway that, in sensitive cell types, ultimately ends in apoptotic cell death. The Fas/FasL pathway is probably best recognized for its role in the downregulation of expanded clonal T lymphocyte populations. In this system, Fas is upregulated within a few hours of T cell activation (Miyawaki et al., 1992, J. Immunol. 149:3753-3758). Several days later, Fas becomes functional and if the cells continue to be stimulated through the antigen receptor, FasL is also upregulated (Owen-Schaub et al., 1992, Cell. Immunol. 140:197-205; Suda et al., 1993, Cell 75:1169-1178), and the majority of activated cells undergo apoptosis, allowing the immune system to return to its normal resting size and repertoire.
In related U.S. Pat. No. 5,759,536, the present inventors disclosed that a factor released by testicular Sertoli cells, which is responsible for the protection of the intratesticular islet allografts and xenografts against rejection, is the Fas ligand. U.S. Pat. No. 5,759,536 disclosed the use of Fas ligand to suppress graft rejection, to suppress T lymphocyte-mediated disease, and to suppress T lymphocyte-mediated disease recurrence. Fas ligand mediates its effect by interacting with Fas. As discussed in detail in U.S. Pat. No. 5,759,536, a major problem associated with transplantation of any tissue is immune-mediated graft rejection in which the recipient's T-lymphocytes recognize donor histocompatibility antigens as foreign. Current regimes for transplanting many tissues and organs require lifelong administration of immunosuppressive drugs. These drugs have serious side-effects and can cause increased susceptibility to infection, renal failure, hypertension, and tumor development. Fas ligand/Fas-mediated apoptosis provided a novel solution to these problems.
In addition to regulating immune responses, Fas and its ligand are likely to play a role in other systems as well. For example, the testes and placenta, both of which are known to be immune-privileged tissues, express FasL (Xerri et al., 1997, Mol. Pathol. 50:87-91). Additionally, Fas and FasL have been found to be coexpressed in a few epithelial tissues that are marked by apoptotic cell turnover, such as the uterus and prostate (Leithauser et al., 1993, Lab. Invest. 69:415-429; French et al., 1996, J Cell. Biol. 133:335-343; and Xerri et al., 1997, supra). Both of these tissues are steroid-dependent and undergo apoptosis within 24-48 h after hormone depletion (Issacs et al., 1992, J. Androl. 19:457-464; Rotello et al., 1992, Am. J. Pathol. 140:449-456). Interestingly, the apoptosis that occurs in response to steroid depletion has recently been shown to require sufficient Fas expression (Suzuki et al., 1996, EMBO. J. 15:211-215; Suzuki et al., 1996, Oncogene 13:31-37). These data support a role for the Fas signaling pathway in the normal renewal of the uterine and prostatic epithelium.
In addition to the in vivo data above, several laboratories have recently demonstrated a potential role for Fas-dependent apoptosis in human prostate cancer (PC) cell lines (Rokhlin et al., 1997, Cancer Res. 57:1756-1758; Uslu et al., 1997, Clin. Cancer Res. 3:963-972; Hedlund et al., 1998, Prostate 36:92-101). Although Fas expression has proved to be a common feature of the cell lines studied, contradictory results were reported with regard to their apoptotic potentials. This may be due to the use of different agonistic anti-Fas antibodies among laboratories, or to the different experimental conditions that were employed. Taken as a whole, these studies indicated that the apoptotic potential of cells expressing Fas may not be sufficient to enable the widespread use of Fas ligand as a therapeutic agent.
In addition, the ability to produce Fas ligand in a form and quantity which is readily useable for both in vitro and in vivo scientific and clinical protocols has been a problem experienced by several researchers. In particular, production of viral vectors encoding apoptosis-inducing proteins such as Fas ligand has met with limited success, due to massive death of the cells used to package and/or deliver such vector and/or unsuitably low viral production (Larregina et al., 1998, Gene Therapy 5:563-568; Muruve et al., 1997, Hum. Gene. Ther. 8:955-963; Arai et al., 1997, PNAS USA 94:13862-13867; Kang et al., 1997, Nature Med. 3:738-743). Such problems are particularly apparent when efforts have been made to scale up production of the vector. This problem of autocrine regulation can be generally extended to other apoptosis-inducing proteins. Therefore, although Fas ligand and/or other apoptosis-inducing proteins appear to be ideal candidates for various therapeutic protocols, including suppression of graft rejection, suppression of T-lymphocyte-mediated disease, and treatment of cancers, unexpected problems with the production and use of such proteins have hindered efforts to design useful therapeutic strategies using such agents.
Therefore, there is a need in the art for a safe and effective agent, such as a construct encoding Fas ligand and/or other apoptosis-inducing proteins, which is capable of inducing apoptosis in a desired target cell.